Rapid 65-min SYBR-Green PCR Assay for Carbapenem Resistant <i>Klebsiella</i> and <i>Acinetobacter</i> Detection

This study developed a rapid and reliable SYBR-Green semiplex PCR assay for simulta-neous detection of major carbapenem resistance genes in Klebsiella pneumoniae and Acinetobacter baumannii. Two primer sets were used: one to detect blaKPC, blaNDM-1, and blaOXA-48 genes in K. pneumoniae and blaOXA-23 in A. baumannii, and another to amplify conserved 16S rRNA gene regions as internal controls. The intra- and inter-assay coeffi-cient of variation ranged from 0.03% to 3.8%. Standard curves exhibited excellent linearity across six logarithmic scales (101-106 DNA copies/mu L), with detection limits of 10-102 DNA copies/mL. Melting temperatures (Tm) were: 88.85 degrees C (KPIC), 90.65 degrees C (NDM-1), 89.45 degrees C (KPC), 84.23 degrees C (OXA-48), 87.81 degrees C (OXA-23), and 80.67 degrees C (ABIC). The SYBR-Green Semiplex PCR assay offers a rapid (65 min turnaround), cost-effective, and sensitive method for early detection of carbapenem-resistant pathogens, enabling timely targeted therapy and improved infection control by potentially reducing empirical antibiotic use before culture confirmation.